No abstract

SummaryWound healing crucially relies on the mechanical activity of fibroblasts responding to TGFb1 and to forces transmitted across focal adhesions. Integrin-linked kinase (ILK) is a central adapter recruited to integrin b1 tails in focal adhesions mediating the communication between cells and extracellular matrix. Here, we show that fibroblast-restricted inactivation of ILK in mice leads to impaired healing due to a severe reduction in the number of myofibroblasts, whereas inflammatory infiltrate and vascularization of the granulation tissue are unaffected. Primary ILK-deficient fibroblasts exhibit severely reduced levels of extracellular TGFb1, a-smooth muscle actin (aSMA) production and myofibroblast conversion, which are rescued by exogenous TGFb1. They are further characterized by elevated RhoA and low Rac1 activities, resulting in abnormal shape and reduced directional migration. Interference with RhoA-ROCK signaling largely restores morphology, migration and TGFb1 levels. We conclude that, in fibroblasts, ILK is crucial for limiting RhoA activity, thus promoting TGFb1 production, which is essential for dermal repair following injury.

Pseudoachondroplasia and multiple epiphyseal dysplasia are two dominantly inherited chondrodysplasias associated with mutations in cartilage oligomeric matrix protein (COMP). The rarely available patient biopsies show lamellar inclusions in the endoplasmic reticulum. We studied the pathogenesis of these chondrodysplasias by expressing several disease-causing COMP mutations in bovine primary chondrocytes and found that COMP-associated chondrodysplasias are not exclusively storage diseases. Although COMP carrying the mutations D469⌬ and D475N was retained within the endoplasmic reticulum, secretion of COMP H587R was only slightly retarded. All pseudoachondroplasia mutations impair cellular viability and cause a disruption of the extracellular matrix formed in alginate culture irrespective of the degree of cellular retention. The mutation D361Y associated with the clinically milder disease multiple epiphyseal dysplasia gave mild retention and limited matrix alterations, but the transfected cells showed normal viability. The effect of mutated COMP on matrix formation and cell-matrix interaction may be a major element in the pathogenesis of COMP-associated chondrodysplasias.

Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with a TSP-4 specific antiserum, but also with an antiserum to COMP, when submitted to SDS-PAGE under nonreducing conditions. Two-dimensional SDS-PAGE (nonreducing followed by reducing conditions) and immunoprecipitation as well as MALDI-TOF mass spectrometry analysis showed that TSP-4 and COMP are both present in equine tendon and cannot be separated under nonreducing conditions despite significant differences in subunit size. This suggests that they are connected via disulfide bridges into heterooligomers.

The influence of a coordinated π-radical on the spin crossover properties of an octahedral iron(II) complex was investigated by preparing and isolating the iron(II) complex containing the tetradentate N,N'-dimethyl-2,11-diaza[3.3](2,6)pyridinophane and the radical anion of N,N'-diphenyl-acenaphtene-1,2-diimine as ligands. This spin crossover complex was obtained by a reduction of the corresponding low-spin iron(II) complex with the neutral diimine ligand, demonstrating that the reduction of the strong π-acceptor ligand is accompanied by a decrease in the ligand field strength. Characterization of the iron(II) radical complex by structural, magnetochemical, and spectroscopic methods revealed that spin crossover equilibrium occurs above 240 K between an S=1/2 ground state and an S=3/2 excited spin state. The possible origins of the fast spin interconversion observed for this complex are discussed.